There have been recent advances in the use of retrovirus-derived vaccines for the treatment of HIV. Specifically, a formalin-inactivated whole HIV vaccine has been developed which has conferred protection in Macaques. Immunization with vaccines potentiated with albumin has resulted in the protection from clinical disease in eight out of nine monkeys challenged with infectious doses of HIV. Notably, protection could be achieved even in cases where entry of viruses is not prevented, suggesting that it may not be necessary to completely block infection in order to have a successful vaccine.
Whole killed HIV vaccines have also been beneficial in the treatment of chimpanzees who were previously infected by HIV. These chimpanzees appear to have cleared the HIV infection in their blood streams following the vaccinations. Post-exposure immunization in humans has also been studied. These tests suggest that immunization may be used to protect humans from HIV infections, and also to treat humans who have already been infected with the virus. However, whole virus vaccines may contain infectious particles. As a result, it may be safer to use essential components of the virus to confer protection. Epitopes of the virus are one example of a safer, essential component of the virus. More recent studies have confirmed that partial protection from infection can be achieved also by gp120 and gp160 derived vaccines. (Desrosiers, R. C. et al. Proc. Nat'l. Acad. Sci. USA, 86:6353 (1989), Kestler, et al. Science, 248:1109 (1990); Murphey-Corb, M. Science, 246:1293 (1989)).
It has long been recognized that peptide epitopes of amino acids conjugated to immunogenic carriers can elicit high levels of high affinity antipeptide antibodies. (See Talwar, G. P., Bloom, B. et al., "Biological and Clinical Aspects of Reproduction", Exceptor Med. Series 394:2224-2232 (1987), in which the beta chain of human chorionic gonadotropin was conjugated to tetanus toxoid to produce an antifertility vaccine.)
The carriers to which peptides are conjugated in this invention have all been used as immunogenic carriers in animals, and some have been used in humans. By way of example, the purified protein derivative (PPD) of tuberculin from Mycobacterium tuberculosis, which is the preferred carrier of the invention, is a unique immunologic reagent, because virtually everyone in the world with a functional immune response who has been exposed to BCG or M. tuberculosis infections will have a T-cell mediated, delayed-type hypersensitivity response to minute amounts of PPD. Tuberculin-PPD conjugates have been utilized in the past. Mice pre-sensitized or "primed" with BCG can produce high levels of antibodies to peptide or carbohydrate epitopes conjugated to PPD. Of particular interest are studies on the NANP repeating epitope of the P. falciparum circumsporozoite antigen, which is immunogenic in only two strains of mice. Conjugating the NANP repeating peptide to PPD elicits the production of antibody titers greater than 1:1000 in genetically non-responder strains to the NANP epitope. This degree of response is comparable to that seen in responder strains given the peptide conjugate in complete Freund's adjuvant. (See Lussow et al., "Use of Tuberculin Purified Protein Derivative-Asn-Ala-Asn-Pro Conjugate in Bacillus Calmette-Guerin Primed Mice Overcomes H-2 Restriction of the Antibody Response L and Avoids the Need for Adjuvants," Proc. Nat'l Acad. Sci. USA 87 (1990)).
Pseudomonas aeruginosa exotoxin A (toxin A) has been used effectively as a carrier in conjugate vaccines. Conjugates made with this carrier have higher immunogenicity, especially when coupled with the recombinant protein R32 to create an immune response against the sporozoite stage of Plasmodium falciparum. Pseudomonas aeruginosa exotoxin A may be purified from the supernatant of fermentor-grown cultures of Pseudomonas aeruginosa
103. Toxin A has been classified as a superantigen based upon results in animals. Toxin A can be completely and irreversibly detoxified by covalent coupling to adipic acid dihydrazide (ADH), a 4 carbon spacer molecule. This step destroys the ADPR-transferase activity of the toxin molecule, hence rendering it nontoxic. The non-reacted hydrazide group can be used to covalently couple haptens to toxin A.
To date, the following haptens have been coupled to toxin A by the inventors by the use of ADH and carbodiimide as a coupling agent: (1) small molecular weight polysaccharides from P. aeruginosa and Escherichia coli; (2) the immunodominant (NANP).sup.3 repeat from Plasmodium falciparum circumsporozoite; and (3) a recombinant protein, termed R32LR, which contains multiple NANP and NVDP repeats from P. falciparum.
Approximately 5,000 subjects have been immunized by the inventors with toxin A-containing vaccines produced by the inventors. As much as 400 mg of toxin A have been administered per dose, with multiple (3) doses given to subjects. These vaccines have been very well tolerated. Mild to moderate, transient local reaction occur in 0.25% of vaccines. Systemic reactions occur in 0.1-2%. Abnormal blood chemistries have not been associated with these vaccines.
Keyhole Limpet Hemocyanin (KLH) is a high molecular weight protein which is purified from megathura crenulata. KLH has many available primary amines from lysine residues which facilitate protein conjugation. KLH is highly immunogenic, and because of its availability of primary amines, is ideal for protein conjugation.
Tetanus and diphtheria toxoids have also been used successfully as protein carriers. Diphtheria toxoid has been used with a synthetic 31 amino acid N-terminal peptide. Both tetanus toxoid and diphtheria toxoid have proved to be effective carriers in humans for the poorly immunogenic carbohydrate antigen of Hemophilus influenza b.
The recombinant core antigen of hepatitis B has the capability of self-assembling into 27 millimeter particles which are highly immunogenic in experimental animals. These HBV core particles may be conjugated directly with peptides, using recombinant DNA technology. Fusion proteins can be produced between the HBV core antigen and defined sequence peptides with high epitope density, which lead to high titer antibodies, as well as to long lasting neutralizing antiviral immunity. Hepatitis B core antigens and self-assembled HBc-HIV peptide fusion protein may be used as protein carriers.
It has been established that the major antigenic component of the mycobacterial cell wall is a protein which consists of a polypeptide monomer of between 10 and 16 Kd, the amino terminal sequence of which reveals that it is related to the GroES heat-shock protein present in many bacteria. It has been indicated that the major antigenic component of mycobacteria recognized by CD4+ T-cells is associated with the cell wall. BCG cell wall (purified) may be used as a protein carrier. BCG may also be used to prime animals or humans prior to vaccination. BCG priming enhances the humoral and cellular responses induced by vaccination.
Currently, the only adjuvant licensed for use in man is alumina. In the present invention, alumina may be used with the conjugates which include the carriers Pseudomonas aeruginosa exotoxin A, KLH, and tetanus and diphtheria toxoids. Aluminum-based gels such as Al(OH).sub.3 and AlP0.sub.4, as well as liposomes may be used as adjuvants with the carrier Pseudomonas aeruginosa exotoxin A in the present invention. PPD conjugates can be given in saline with no further adjuvants to tuberculin-positive subjects. For the BCG cell wall and HBc antigen conjugates, it is likely that adjuvants would be required. Ribi adjuvant containing trehalose dimycolate and 2% squalene may be used as an adjuvant. Alternatively, if studies on the long-term safety of incomplete Freund's adjuvant or ISCOM's adjuvant indicate their safety and efficacy in humans, these adjuvants may be used. Microencapsulation technology using polyactide/polyglycolide biodegradable polymers may also be used as an adjuvant.
Because a significant amount of HIV-1 transmission occurs from cell to cell (see McCune, "HIV-1: The Infective Process in vivo", Cell, Vol. 64, pp. 351-363 (1991)), neutralizing antibodies alone cannot prevent clinical infection. Furthermore, since the majority of HIV-1 transmission occurs via the mucosal route, effective mucosal immunity is necessary for protection from HIV-1 infection. The effectiveness of an HIV-1 vaccine depends upon its capacity to induce HIV-1 specific cell mediated immunity and humoral immunity in both serum and in the mucosa.
To date, no preventive vaccine has been reported which induces humoral, cellular and mucosal immune response. Hence, it is desirable to develop a vaccine which induces antibody (serum and mucosal) as well as PND-specific T cell (CTL) response after immunization therewith.
Current results with post-infection HIV-1 recombinant gp120 and gp160 (Salk J, et al. Science 1993; 260: 1270-72; Redfield R R, et al. N Engl J Med 1991; 324: 1667-84; Valentine F T, et al. J Infect Dis 1996; 173: 1336-46; Eron J J, et al. Lancet 1996; 348: 1547-51; Haynes B F Lancet 1996; 348:933-37; Haynes B F Lancet 1996; 348: 1531-2) vaccines are discouraging. (Redfield R R, et al. N Engl J Med 1991; 324: 1667-84; Valentine F T, et al. J Infect Dis 1996; 173: 1336-46; Eron J J, et al. Lancet 1996; 348: 1547-51). In light of the enormous turnover of HIV-1 virions and CD4 cells (Saag M S, et al. Nat Med 1996; 625-29; Mellors J W, et al. Ann Intern Med 1995; 122: 573-79; Ho Dd, et al. Nature 1995; 373: 123-126; Fauci A S Nature 1996; 384: 529-33) it was suggested that the prospect of ever inducing a more effective anti-HIV-1 immunity was slim in an immune system that is already working overtime. (Haynes B F Lancet 1996; 348:933-37; Haynes B F Lancet 1996; 348: 1531-2; Haynes B F, et al. Science 1996; 271: 324-8).
In contrast to this bleak outlook are observations of immune responses in long term non-progressors that are absent or decreased in rapid progressors (Haynes B F Lancet 1996; 348:933-37; Haynes B F Lancet 1996; 348: 1531-2; Haynes B F, Science 1996; 271: 324-8) and recent studies in infants have indicated the existence of abortive infections. (Bryson Y J, N Engl J Med 1995; 332: 833-8; Roques P A, AIDS 1995; 9: F19-26.) The development of our vaccine was based on studies showing a correlation between high affinity antibodies to gp120 or to the V.sub.3 loop and reduced matemofetal HIV-1 transmission. (Goedert J J, Lancet 1989; ii: 1351-53; Rubinstein A, et al. AIDS 1995; 9: 243-51). The V.sub.3 loop is known to participate in vital viral properties such as cell tropism and cell fusion. Antibodies to the V.sub.3 loop decline with disease progression, while antibodies to whole gp160 remain stable. (Fenouillet E, Clin Exp Immunol 1995; 99: 419-24). Guinea pig immune sera against a BCG vector secreting the V.sub.3 -Primary Neutralizing Domain (PND) blocked HIV-1 infection in SCID/hu mice (Honda M, et al. Proc Natl Acad Sci USA 1995; 92: 10693-97) and a monoclonal antibody to the V.sub.3 loop protected chimps against HIV-1 infection. (Emini E A, et al. Nature (London) 1992; 355: 728-3018).
Thus, there remains a need for the discovery and development of peptide carrier conjugate vaccines capable of inducing prolonged antibody immune response. There is an additional need for vaccines which are capable of inducing a serum humoral immune response, mucosal humoral immune response and the production of cytotoxic lymphocytes. In addition, there is a great need for the discovery and development of an effective method of treatment and prevention of HIV infection which will reduce the viral load in a subject, thereby preventing or limiting the progression of the disease.